Plant germplasm resistant to rna viruses

ABSTRACT

Disclosed is a dsRNA construct used to silencing specific eukaryotic translation initiation factor in plants to produce a plant resistant to viruses such as Potyviruses, Luteoviruses, and Furoviruses. More specifically, the plant would be resistant to viruses such as Wheat streak mosaic virus, Triticum mosaic virus, Soil bourne mosaic virus, or Barley yellow dwarf virus. Also disclosed are non-transgenic wheat plants having the genes for eIF(iso)4E-2 or eIF4G silenced.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional application of U.S. applicationSer. No. 15/875,168 filed Jan. 19, 2018, which is a divisionalapplication of and claims priority to U.S. application Ser. No.14/494,661, filed Sep. 24, 2014, which itself claims priority under 35U.S.C. § 119(e) to U.S. Provisional Ser. No. 61/882,116, which was filedon Sep. 25, 2013, the entirety of all of which is hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention is related to silencing specific eukaryotictranslation initiation factors in plants to produce a plant resistant toviruses such as Potyviruses, Luteoviruses, and Furoviruses. Morespecifically, the plant would be resistant to viruses such as Wheatstreak mosaic virus, Triticum mosaic virus, Soil bourne mosaic virus, orBarley yellow dwarf virus.

BACKGROUND OF INVENTION

Fire, et al. (U.S. Pat. No. 6,506,559) discloses a process ofintroducing RNA into a living cell to inhibit gene expression of atarget gene in that cell. The RNA has a region with double-strandedstructure. Inhibition is sequence-specific in that the nucleotidesequences of the duplex region of the RNA and of a portion of the targetgene are identical. Specifically, Fire, et al. (U.S. Pat. No. 6,506,559)discloses a method to inhibit expression of a target gene in a cell, themethod comprising introduction of a double stranded ribonucleic acidinto the cell in an amount sufficient to inhibit expression of thetarget gene, wherein the RNA is a double-stranded molecule with a firstribonucleic acid strand consisting essentially of a ribonucleotidesequence which corresponds to a nucleotide sequence of the target geneand a second ribonucleic acid strand consisting essentially of aribonucleotide sequence which is complementary to the nucleotidesequence of the target gene. Furthermore, the first and the secondribonucleotide strands are separately complementary strands thathybridize to each other to form the said double-stranded construct, andthe double-stranded construct inhibits expression of the target gene.

To utilize RNA interference as a method to regulate gene expression forcontrol, a specific essential gene needs to be targeted. Coordinatedgene expression requires factors involved in transcription andtranslation. Translation initiation coordinates activities of severaleukaryotic initiation factors (eIF) or proteins which are classicallydefined by their cytoplasmic location and ability to regulate theinitiation phase of protein synthesis. One of these factors, the eIF4Fcomplex involves the expression of two proteins EIF(iso)4E-2 and EIF4G.

Disclosed in Caranta et al. (US Pat. No. 7,919,677) is a method toobtain Potyvirus resistant plants having mutations in an eIF4Etranslation factor region. Additionally, disclosed in Jahn et al. (U.S.Pat. No. 7,772,462) is silencing of translation initiation factor eIF4Efrom Capsicum annuum imparted virus resistance to transgenic plants.Given the interest and showing of viral resistance to poyviruses relatedto eIF4 region, there is a need in the art to determine whethertargeting eIF(iso)4E-2 or eIF4G via RNA interference induces viralresistance in vial susceptible plants.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention together with the above and other objects andadvantages may best be understood from the following detaileddescription of the embodiment of the invention illustrated in thedrawings, wherein:

FIG. 1A and FIG. 1B depicts a vector map for a CP fragment cloned intopANDA mini vector by means of homologous recombination via LR clonase.

BRIEF DESCRIPTION OF THE SEQUENCES

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions, which form apart of this application.

SEQ. ID. NO:1: ATGGCAGAGGTCGAAGCTGCGCTCCCGGTGGCGGCGACAGAGACCCCGGAGGTCGCCGCCGAGGGCGACGCGGGTGCGGCCGAGGCGAAGGGGCCGCACAAGCTGCAGCGGCAGTGGACCTTCTGGTACGACATCCAGACCAAGCCCAAGCCCGGCGCCGCCTGGGGCACCTCGCTCAAAAAGGGCTACACCTTCGACACCGTCGAAGAGTTCTGGTGCTTGTATGATCAGATTTTCCGTCCGAGTAAGCTGGTAGGAAGTGCTGATTTTCATTTATTCAAGGCTGGGGTAGAACCAAAGTGGGAAGATCCAGAGTGCGCAAATGGAGGCAAATGGACTGTGATATCTAGCAGGAAGACCAATCTTGATACCATGTGGCTTGAAACGTGTATGGCTCTGATTGGAGAGCAGTTCGATGAAAGCCAGGAAATTTGTGGTGTTGTTGCTAGTGTCCGCCAGAGACAGGATAAGCTTTCATTATGGACTAAGACTGCCAGTAACGAAGCTGTTCAGGTGGACATTGGCAAGAAATGGAAGGAGGTTATTGACTACAATGATAAGATGGTCTACAGCTTCCACGATGACTCAAGAAGTCAGAAACCAAGCAGAGGTGGACGA TACACCGTCTAAcorresponds to the cDNA of eIF(iso)4E-2.

SEQ. ID. NO: 2: MAEVEAALPVAATETPEVAAEGDAGAAEAKGPHKLQRQWTFWYDIQTKPKPGAAWGTSLKKGYTFDTVEEFWCLYDQIFRPSKLVGSADFHLFKAGVEPKWEDPECANGGKWTVISSRKTNLDTMWLETCMALIGEQFDESQEICGVVASVRQRQDKLSLWTKTASNEAVQVDIGKKWKEVIDYNDKMVYSFHDDSRSQKPSRGGRYTV corresponds to the amino acidsequence encoded by SEQ. ID. NO:1.

SEQ. ID. NO: 3:ATGGGGCATCAAGGACAAACCATGATGTATCCGTCTGTTGCTCATCCAATCCCTCCTCAACTGGGCAATGTTAATTTGAACATGGCTTCACAGTATCCTCAGCAACAGCAGAATAAGCTTGTTGCTCCTCGAAAGAGCAGTAATATCAAAATTACTGATCCAAACACTAACAAAGAAGTGGTTCTTGGGCGGCCTTCACCTAATGTAGCAGTACAACCGCAGCAAGTCAGTGGTGTTGCAACTCAGCCTATGGTTTACTATACTAATCCACAGCAGACCTCGTATAACCAGTCAGGCACGTATTACTCCGGCACTGCTGGTGTTGTTCCCACTGGATCACAGGGCAGGTTTGGTTATCCTGCCACTCAAGCTGGTCAATCAATTCCTTTCATGAACCCTTCTATGTCAAATACTGTTCCTGCCAGCCACAAGGACAACATAGCTGGGCCTGCACCATCTGGTCAGTCCCAACTCATAGGCAAACCACAAGGTGGGTTGCACATGGAGAAACCTGTTCCCTCGGTCAAGATAAGTATGCCTGCAGGTAGATCAGACGCTTCTAAATTCGGGGTCGCTGACCATGCGGTACAACATCGACAAAAGGATAATGAAGTTATTTCTGGTGCTATGGTTTCGAATAAACCAGTTAGTGAGAAGGAGAGCAAGGCACCATCTATCCCAGAGAAGCACTCCAAGGAAAGTAAAGCACCATCTGCCGTGGAGAAGCATCCCACTGCGGTGACTCAACCTTTACCGATTCAAGCTGCAAAGCCAGAAACTGATGCAGCGACTGCAAATTCACCCTCATTCTTGACCGGAGCTGATGAAAAGAAAGAATCCCTTCCAATGACTGATTCACTTAAGGATAACAAGAAAAATGCAACTAGAAATGACACAAAGAATTTGCCGCAACAACCTCAGTCTGCTTCCCCTGCCGAAGAGTTGAAGGGGCAAACTTCTGTGAAGCTTGGTGATGATGTGGTTGGTCACATGGAAACCAAGAGCTTCGATAGTGAAAAGGTGGATTTAACCAGCAAGGTTTCAGGCTTAACATCAGCAACATCTGAAAGTAGTATTTCTCCTATTCTTGGTAAAAGTGAAGCTGACAGCACATCAGTAGATGCTGCTGATGTTCCTGCCATGGTAATCAGCTCTGCAAAATTGTCCTCTGCGAGCACTGGGGAGCCCCAAGCAGTAGAAAGCTTAGGTGTTGCTGCTGTTAAATCTAAGGAGATTGAAATAACTCACCAAATTTCACCTGAATCTAGTGATGGCAAAATTATGTCTGATTCTACTGAAAATGAATCACATGACTTCACGGTGGACTTGGCTGAGCAGGCATCATTGGCAACTTCAAAGCCTGGTAATTCAGATGCAACATCTTTTGTAACTGACCCGCAAGAGCTACCCAAGGAGTGCACAACATCTGTACCGGAGGACCACAGTTTGATGAATACATCACATAATAAGGATACCCAAACTTTATCAGCTTCTGTGGATGCCAGCGATGTGTCTGAGGTCAATTCTGGAACCTCATCAGAGTCTACCAGCCAAAGTACCAACGATAAAGATATCAGAAGTAGCATTCAGGAAACTGGATTAGCTGTTTCTGGTATTACTCCTGGCATGTTGCCTGTGAATCATTCAGTTGCATCTGAGGGGCAAGTCAAACATGCAGATGGAGCGAAGGATGAGTCTAGTACTGAGCAATCAAGTGCCGTACCAACAGGTTCTGTTAGACCCTTATCAAGGGAAAAACCTACTGCAGAGCTTGCCCGAACAAAGTCTACAGCTGGGAGAAAGAAGAAACGGAAGGAAATGCTTTCAAAAGCTGATGCTGCTGGGAGCTCAGATCTGTACAATGCATACAAAGGACCACAAGAACAGTCTGAGAGTGTTGCCACATCAGACGGTGCTGATAGTTCTTCAACAGTCGACGGGACACATGTGCTGCCTGAGGAATCAGAAAGGGAGGTGATGTGTGAGGACGATGGAAAGAAAAAAGTTGAGCCGGATGATTGGGAAGATGCAGCAGACATGTCTACTCCAAAGCTGCAAAGTTCGGACTCTGGAAACCAGGCTAGTGCAGTTCAATTGCCAGATTCTGATATGACTGAAGCTAATGGCCGAAAGAAATATTCTCGTGATTTTCTTCTAACTTTTGCACATCAGTATTCTAGTCTTCCTGTTGGCATCCGGATGGATACTGTCACTAGTACGCTATTCAAAGATTTGGCAGGAAAATCCTATGTTATTGATCGGGAACCTCACCCAAGTTCTGCAAGGGGATCTGATAGACCAACATCTCGCGGTGATCGCCGTGGTCCTGCTATGGATGATGATAAGTGGTTAAAATCAGGTGTTCCTTACAGTCCTAACCGTGATGCCCACATGGACTTGACAAACGGCCCAGCAATTAATTACCGTGGCGGCCCAGGAGGCGCTCATGGTGTTCTGAGGAATCCACGTGGTGCACTCCTTGTGGGACCACAATCCAATGCTCCTCAAGTACCCCGCAGTGGCTCTGATGCAGATAGATGGCAGCAAAAGGGTCTGATCCCATCTCCTGTTACACCCATGCAAGTAATGCACAAAGCCGAGAAAAAGTATGTTGTCGGCAAAGTTTCTGATGAGGAGCAGGCAAAGCAGAGGCAGCTGAAAGCCATTCTGAATAAACTGACCCCACAAAACTTTGACAAGCTTTTTGAACAAGTGAAAGAGGTGAACATTGACAATGTATCAACTCTTACTGGGGTGATTTCACAGATATTTGACAAAGCTTTGATGGAACCAACTTTCTGTGAAATGTATGCCAACTTCTGTTCCCATTTGGCTGGTGCCCTGCCAGACTTTAGTGAGGACAATGAAAAGATTACATTCAAGAGACTGCTATTGAACAAGTGCCAAGAGGAGTTTGAGAGGGGAGAAAGAGAAGAAGCTGAAGCAGATAAAACGGAGGAGGAAGGTGAGATTAAGCAAACGAAAGAGGAAAGGGAAGAAAAGAGAGTTAAAGCTCGAAGGCGCATGCTGGGTAATATTAGATTGATTGGAGAATTGTACAAAAAGAGGATGTTGACAGAGCGCATCATGCATGAATGCATCAAAAAATTGTTGGGAAATTATCAGAATCCAGATGAGGAGAACATTGAAGCACTATGCAAATTGATGAGTACAATTGGAGAGATGATAGATCATCCCAAGGCTAAGGAACATATGGATGCNTATTTTGATAGAATGCGCAACCTGTCGACCAGTCAACTGATATCTTCCCGTGTTAGATTCCTGCTCAGAGATTCAATCGATCTCAGGAAGAACAAATGGCAGCAAAGGCGTAAAGTGGATGGCCCCAAGAAGATTGATGAGGTTCACAGGGATGCAGCTCAGGAAAGACATGCTCAATCGAGTAGGTCTCGTGGTCCAGTCGTTAGTTCTCTTCCAAGAAGAGGGGCACCCTCTATGGATTACGGCTCCCGTGGCTCAGCAGCACCATTGGTATCTCCAGGTCCTCAGCAACGAGGGCGTGGATTTGGTAATCAAGATATTCGGTATGAGCAGGAAAGGCATCAGTTTGATAGAACTGTTCCCCTTCCCCAGCGTTCTGTAAAGGACGAAGCTATCACTCTTGGACCACAAGGTGGCCTAGCTAGGGGTATGTCTTTAAGAGGGCAGCCACCGGTATCAAATTCTGAACTTCCTAGTGTTGTTGACCAGCGCAGGATTGTATCTGGTCCTAATGGGTACAATTCTGTGCCTTCAACAACAAGAGAAGACACTAGCTCTAGAATTCCAGATCGATTTTCTGGGAGAATAGCACCTGCTGCACAATCTGCTAGTTCTTCACACAGACCTGCCAGCCAGGAGGGTCGTTCAGGAAATAAATCATACTCTGAGGAGGAATTGAGAGAGAAATCTATTGCAACCATCCGGGAATATTATAGTGCGAAAGATGAAAAGGAAGTTGCATTGTGTATTGAGGAGTTGAATGCTCCGAGCTTCTATCCTTCTCTTGTATCACTTTGGGTAAATGATTCCTTTGAGAGGAAAGATATGGAAAGAGAGTTGTTGGCAAAGCTCTTTGTCGGGCTTTACAATGGTGGATATAATTTATTGAGCAAGCCTCAGCTCATTGAGGGGCTTTCATCCGTTCTTGCTTCATTGGAGGATGCTCTAAGTGATTCTCCAAGAGCGGCAGAGTATCTTGGACGTCTTCTTGCAAGGTTTGTGGTGGAGAAGATACTGGTTTTGCAAGACGTAGGTAAATTGATTGAAGAAGGCGGAGAGGAGCCTGGACACCTTGTGCAGGAAGGCATCGCAGCTGATGTCCTTGGCGCAGTCTTGGAGTGGATCAGAACAGAAAAGGGGGATTCCTTCTTGAAGGAGGCCAAGACAAGCTCCAATCTCAAGTTGGAGGATTTCAGACCGCAGCATCTTAAGAGGTCAAAGTTGGATGCCTTCATGTTGACTTAA corresponds to thecDNA of eIF4G.

SEQ. ID. NO: 4: MGHQGQTMMYPSVAHPIPPQLGNVNLNMASQYPQQQQNKLVAPRKSSNIKITDPNTNKEVVLGRPSPNVAVQPQQVSGVATQPMVYYTNPQQTSYNQSGTYYSGTAGVVPTGSQGRFGYPATQAGQSIPFMNPSMSNTVPASHKDNIAGPAPSGQSQLIGKPQGGLHMEKPVPSVKISMPAGRSDASKFGVADHAVQHRQKDNEVISGAMVSNKPVSEKESKAPSIPEKHSKESKAPSAVEKHPTAVTQPLPIQAAKPETDAATANSPSFLTGADEKKESLPMTDSLKDNKKNATRNDTKNLPQQPQSASPAEELKGQTSVKLGDDVVGHMETKSFDSEKVDLTSKVSGLTSATSESSISPILGKSEADSTSVDAADVPAMVISSAKLSSASTGEPQAVESLGVAAVKSKEIEITHQISPESSDGKIMSDSTENESHDFTVDLAEQASLATSKPGNSDATSFVTDPQELPKECTTSVPEDHSLMNTSHNKDTQTLSASVDASDVSEVNSGTSSESTSQSTNDKDIRSSIQETGLAVSGITPGMLPVNHSVASEGQVKHADGAKDESSTEQSSAVPTGSVRPLSREKPTAELARTKSTAGRKKKRKEMLSKADAAGSSDLYNAYKGPQEQSESVATSDGADSSSTVDGTHVLPEESEREVMCEDDGKKKVEPDDWEDAADMSTPKLQSSDSGNQASAVQLPDSDMTEANGRKKYSRDFLLTFAHQYSSLPVGIRMDTVTSTLFKDLAGKSYVIDREPHPSSARGSDRPTSRGDRRGPAMDDDKWLKSGVPYSPNRDAHMDLTNGPAINYRGGPGGAHGVLRNPRGALLVGPQSNAPQVPRSGSDADRWQQKGLIPSPVTPMQVMHKAEKKYVVGKVSDEEQAKQRQLKAILNKLTPQNFDKLFEQVKEVNIDNVSTLTGVISQIFDKALMEPTFCEMYANFCSHLAGALPDFSEDNEKITFKRLLLNKCQEEFERGEREEAEADKTEEEGEIKQTKEEREEKRVKARRRMLGNIRLIGELYKKRMLTERIMHECIKKLLGNYQNPDEENIEALCKLMSTIGEMIDHPKAKEHMDAYFDRMRNLSTSQLISSRVRFLLRDSIDLRKNKWQQRRKVDGPKKIDEVHRDAAQERHAQSSRSRGPVVSSLPRRGAPSMDYGSRGSAAPLVSPGPQQRGRGFGNQDIRYEQERHQFDRTVPLPQRSVKDEAITLGPQGGLARGMSLRGQPPVSNSELPSVVDQRRIVSGPNGYNSVPSTTREDTSSRIPDRFSGRIAPAAQSASSSHRPASQEGRSGNKSYSEEELREKSIATIREYYSAKDEKEVALCIEELNAPSFYPSLVSLWVNDSFERKDMERELLAKLFVGLYNGGYNLLSKPQLIEGLSSVLASLEDALSDSPRAAEYLGRLLARFVVEKILVLQDVGKLIEEGGEEPGHLVQEGIAADVLGAVLEWIRTEKGDSFLKEAKTSSNLKLEDFRPQHL KRSKLDAFMLTcorresponds to the amino acid sequence encoded by SEQ ID NO:3.

SEQ. ID. NO: 5: CACCCGCAAATGGAGGCAAATGGACTGT is a forward primer used toamplify a fragment of eIF(iso)4E-2.

SEQ. ID. NO: 6: TCCACCTCTGCTTGGTTTCTGACT is a reverse primer used toamplify a fragment of eIF(iso)4E-2.

SEQ. ID. NO: 7: CACCTCAGCAGCACCATTGGTATCTCCA is a forward primer used toamplify a fragment of eIF4G.

SEQ. ID. NO: 8 GCTCGGAGCATTCAACTCCTCAA is a reverse primer used toamplify a fragment of eIF4G.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is a method of producing a plant germplasm havingresistance to RNA viruses, the method comprises introducing into aparental plant germplasm a chimeric DNA molecule comprising (i) a plantexpressible promoter, (ii) a region which encodes dsRNA for eIF(iso)4E-2or eIF4G which is capable of inhibiting RNA viral replication (iii)plant translation termination signal, b) transforming said parentalplant germplasm, c) generating a plant germplasm from the parental plantgermplasm comprising the chimeric DNA molecule; and d) selecting plantgermplasm obtained from step (c) and identifying germplasm havingimmunity to viral RNA replication. In one embodiment of the invention,also disclosed is a transgenic plant produced by the method disclosedherein. In another embodiment of the invention, disclosed is atransgenic plant produced by the method disclosed where the plant isresistant to RNA plant viruses selected from the group consisting ofPotyviruses, Luteoviruses, and Furoviruses. In yet another embodiment ofthe invention, disclosed is a transgenic plant produced by the methodwherein the plant is resistant to Wheat streak mosaic virus. In yetanother embodiment of the invention, disclosed is a transgenic plantproduced by the method herein where the plant is resistant to Triticummosaic virus. In yet another embodiment of the invention, disclosed is atransgenic plant produced by the method herein where the plant isresistant to Soil bourne mosaic virus. In another embodiment of theinvention, disclosed is a transgenic plant produced by the method hereinwhere the plant is resistant to Barley yellow dwarf virus.

In another embodiment of invention, the expression of transgenes andresistance phenotype remains stable in multiple generations of theprogeny in the transgenic plant produced by the method herein. Inanother embodiment of the invention, the seed of a transgenic plantproduced by the method herein.

In various embodiments of the invention, the method for transformingplant germplasm can be accomplish through a process selected from thegroup consisting of a biolistic particle delivery system,microprojectile bombardment, viral infection, Agrobacterium-mediatedtransformation, and electroporation.

In another embodiment of the invention, the method for producing a plantgermplasm having resistance to RNA viruses is due to inhibitedexpression of an eIF(iso)4E-2 gene encoding a protein comprising SEQ.ID. NO: 2. In yet another embodiment of the invention, the method forproducing a plant germplasm having resistance to RNA viruses is due toinhibited expression of an eIF4G gene encoding a protein comprising SEQ.ID. NO: 4. In another embodiment of the invention, disclosed is atransgenic plant having resistance to RNA viruses where said transgenicplant comprises a chimeric DNA molecule which encodes a double strandedRNA molecule for eIF(iso)4E-2 or eIF4G.

Disclosed herein is a nucleic acid construct comprising a nucleotidesequence of SEQ. ID. NO: 1 or an antisense sequence corresponding toSEQ. ID. NO: 1, wherein the construct is operably linked to a promoterthat drives expression in a plant cell. Also disclosed is a vectorcomprising of the nucleic acid SEQ. ID. NO: 1. In an embodiment of theinvention is a transgenic plant having stably incorporated in its genomethe nucleotide sequence of SEQ. ID. NO:1.

Disclosed herein is a nucleic acid construct comprising of thenucleotide sequence of SEQ. ID. NO: 3 or an antisense sequencecorresponding to SEQ. ID. NO: 3, wherein the construct is operablylinked to a promoter that drives expression in a plant cell. Alsodisclosed is a vector comprising the nucleic acid SEQ. ID. NO: 3. In anembodiment of the invention is a transgenic plant having stablyincorporated in its genome the nucleotide sequence of SEQ. ID. NO:3.

Disclosed herein is a non-transgenic plant comprising an eIF(iso)4E-2allele in its genome, whereby the eIF(iso)4E-2 allele is an allele whichencodes a protein comprising the amino acid sequence of SEQ. ID. NO: 1,characterized in that said eIF(iso)4E-2 allele comprises one or moremutations in its nucleotide sequence and whereby as a result of said oneor more mutations the plant comprising said mutant allele in its genomehas significant resistance to RNA viruses compared to a plant comprisinga wild type eIF(iso)4E-2 allele in its genome. In an embodiment of theinvention, disclosed is a non-transgenic plant produced by the methoddisclosed where the plant is resistant to RNA plant viruses selectedfrom the group consisting of Potyviruses, Luteoviruses, and Furoviruses.In yet another embodiment of the invention, disclosed is anon-transgenic plant produced by the method wherein the plant isresistant to Wheat streak mosaic virus. In yet another embodiment of theinvention, disclosed is a non-transgenic plant produced by the methodherein where the plant is resistant to Triticum mosaic virus. In yetanother embodiment of the invention, disclosed is a non-transgenic plantproduced by the method herein where the plant is resistant to Soilbourne mosaic virus. In another embodiment of the invention, disclosedis a non-transgenic plant produced by the method herein where the plantis resistant to Barley yellow dwarf virus. In another embodiment of theinvention, the seed of a non-transgenic plant produced by the methodherein.

Disclosed is a non-transgenic plant comprising of an eIF4G allele in itsgenome, whereby the eIF4G allele is an allele which encodes a proteincomprising the amino acid sequence of SEQ. ID. NO: 4, characterized inthat said eIF4G allele comprises one or more mutations in its nucleotidesequence and whereby as a result of said one or more mutations the plantcomprising said mutant allele in its genome has significant resistanceto RNA viruses compared to a plant comprising a wild type eIF4G allelein its genome.

Definitions:

To assist in the understanding of the invention, the following terms, asused herein, are defined below.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

The term “gene” refers to a DNA sequence involved in producing apolypeptide or precursor thereof. The polypeptide can be encoded by afull-length coding sequence or by any portion of the coding sequence,such as exon sequences. In one embodiment of the invention, the genetarget is eIF(iso)4E-2 and eIF4G.

As used herein, the term “isolated” includes a material removed from itsoriginal environment, e.g., the natural environment if it is naturallyoccurring. For example, a naturally occurring polypeptide present in aliving organism is not isolated, but the same polynucleotide orpolypeptide, separated from some or all of the coexisting materials inthe natural system, is isolated. Such polypeptide can be expressed by avector and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment. As used herein, an isolatedmaterial or composition can also be a “purified” composition, i.e., itdoes not require absolute purity; rather, it is intended as a relativedefinition.

The term “oligonucleotide” refers to a molecule comprising a pluralityof deoxyribonucleotides or ribonucleotides. Oligonucleotide may begenerated in any manner, including chemical synthesis, DNA replication,reverse transcription, polymerase chain reaction, or a combinationthereof. The present invention embodies utilizing the oligonucleotide inthe form of dsRNA as means of interfering with a critical developmentalor reproductive process that leads to control. Inasmuch asmononucleotides are synthesized to construct oligonucleotides in amanner such that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points towards the 5′ end of the other,the former may be called the “upstream” oligonucleotide and the latterthe “downstream” oligonucleotide.

The term “primer” refers to an oligonucleotide, which is capable ofacting as a point of initiation of synthesis when placed underconditions in which primer extension is initiated. An oligonucleotide“primer” may occur naturally, as in a purified restriction digest or maybe produced synthetically.

A primer is selected to be “substantially complementary” to a strand ofspecific sequence of the template. A primer must be sufficientlycomplementary to hybridize with a template strand for primer elongationto occur. A primer sequence need not reflect the exact sequence of thetemplate. For example, a non-complementary nucleotide fragment may beattached to the 5′ end of the primer, with the remainder of the primersequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence is sufficiently complementarywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

The term “double stranded RNA” or “dsRNA” refers to two substantiallycomplementary strands of ribonucleic acid. “Identity,” as used herein,is the relationship between two or more polynucleotide sequences, asdetermined by comparing the sequences. Identity also means the degree ofsequence relatedness between polynucleotide sequences, as determined bythe match between strings of such sequences. Identity can be readilycalculated (see, .e.g, Computation Molecular Biology, Lesk, A. M., eds.,Oxford University Press, New York (1998), and Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York (1993),both of which are incorporated by reference herein). While there exist anumber of methods to measure identity between two polynucleotidesequences, the term is well known to skilled artisans (see, e.g.,Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press(1987); and Sequence Analysis Primer, Gribskov., M. and Devereux, J.,eds., M Stockton Press, New York (1991)). Methods commonly employed todetermine identity between sequences include, for example, thosedisclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math. (1988)48:1073. “Substantially identical” as used herein, means there is a veryhigh degree of homology (preferably 100% sequence identity) between theinhibitory dsRNA and the corresponding part of the target gene. However,dsRNA having greater than 90% or 95% sequence identity may be used inthe present invention, and thus sequence variations that might beexpected due to genetic mutation, strain polymorphism, or evolutionarydivergence can be tolerated. Although 100% identity is preferred, thedsRNA may contain single or multiple base pair random mismatches betweenthe RNA and the target gene, provided that the mismatches occur at adistance of at least three nucleotides from the fusion site.

As used herein, “target gene” refers to a section of a DNA strand of adouble-stranded DNA that is complementary to a section of a DNA strand,including all transcribed regions, that serves as a matrix fortranscription. The target gene is therefore usually the sense strand.

The protein EIF(iso)4E-2 is also identified as eukaryotic translationinitiation factor 4E-2, eIF-4E-2, eIF4E-2, eIF-(iso)4F, and eIF-(iso)4Fp28 subunit.

The protein EIF4G is also identified as eukaryotic translationinitiation factor 4G.

The term “complementary RNA strand” refers to the strand of the dsRNA,which is complementary to an mRNA transcript that is formed duringexpression of the target gene, or its processing products. “dsRNA”refers to a ribonucleic acid molecule having a duplex structurecomprising two complementary and anti-parallel nucleic acid strands. Notall nucleotides of a dsRNA must exhibit Watson-Crick base pairs. Themaximum number of base pairs is the number of nucleotides in theshortest strand of the dsRNA.

As used herein, the term “recombinant DNA construct” refer to any agentsuch as a plasmid, cosmid, virus, BAC (bacterial artificial chromosome),autonomously replicating sequence, phage, or linear or circularsingle-stranded or double-stranded DNA or RNA nucleotide sequence,derived from any source, capable of genomic integration or autonomousreplication, comprising a DNA molecule in which one or more DNAsequences have been linked in a functionally operative manner usingwell-known recombinant DNA techniques.

A nucleic acid sequence can be inserted into a vector by a variety ofprocedures. In general, the sequence is ligated to the desired positionin a vector following digestion of the insert and the vector withappropriate restriction endonucleases. Alternatively, blunt ends in boththe insert and the vector may be ligated. A variety of cloningtechniques are known in the art, e.g., as described in Sambrook, J. etal., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., (1989) and Ausubel, F. M. et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y. (1989). Suchprocedures and others are deemed to be within the scope of those skilledin the art.

The vector can be in the form of a plasmid, a viral particle, or aphage. Preferably, as disclosed herein the vector is a bacterial vector.Other vectors include chromosomal, non-chromosomal and synthetic DNAsequences, pET-30a and derivatives of pET-30; bacterial plasmids, phageDNA, baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook, T. et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, Plainview, N.Y., (1989).

“Small interfering RNA” or “siRNA” refers to a short double-strand ofribonucleic acid, approximately 18 to 30 nucleotides in length. The term“RNA interference” or “RNAi” refers to a cellular mechanism for thedestruction of targeted ribonucleic acid molecules. Under endogenousconditions, RNAi mechanism operates when dsRNA is cleaved to siRNA viaan enzyme, DICER. The siRNA is processed to a single strand ofanti-sense ribonucleic acid and coupled with a protein complex namedRISC. The antisense RNA then targets a complementary gene construct,such as messenger RNA that is cleaved by ribonuclease. While theexamples infra discloses constructing dsRNA constructs via enzymatictechniques with the enzyme RNA polymerase, it is contemplated that siRNAcan be constructed via RNA oligonucleotide synthesis such as thosedisclosed in Scaringe, S., Methods Enzymol., 2000, Vol. 317:3 andincorporated herein by reference.

As used herein, “knock-down” is defined as the act of binding anoligonucleotide with a complementary nucleotide sequence of a gene assuch that the expression of the gene or mRNA transcript decreases. In anembodiment, knock-down of a eIF(iso)4E-2 and eIF4G gene in a transgenicplant confers resistance against viral RNA for said transgenic plant.

dsRNA containing a nucleotide sequence complementary to a portion of theeukaryotic translation initiation factor gene, preferably eIF(iso)4E-2and eIF4G. As disclosed herein, 100% sequence identity between the RNAand the target gene is not required to practice the present invention.Thus, the invention has the advantage of being able to tolerate sequencevariations that might be expected due to genetic mutation, strainpolymorphism, or evolutionary divergence. RNA sequences with insertions,deletions, and single point mutations relative to the target sequencemay also be effective for plant resistance to RNA viruses. Thus,sequence identity may be optimized by sequence comparison and alignmentalgorithms known in the art. Thus, the determination of percent identitybetween any two sequences can be accomplished using a mathematicalalgorithm. Non-limiting examples of such mathematical algorithms are thealgorithm of Myers and Miller (1988. CABIOS 4: 11-17), the localhomology algorithm of Smith et al. (1981. Adv. Appl. Math. 2: 482); thehomology alignment algorithm of Needleman and Wunsch (1970. J. Mol.Biol. 48: 443-453); the search-for-similarity-method of Pearson andLipman (1988. Proc. Natl. Acad. Sci. 85: 2444-2448; the algorithm ofKarlin and Altschul (1990. Proc. Natl. Acad. Sci. USA 87: 2264),modified as in Karlin and Altschul (1993. Proc. Natl. Acad. Sci. USA 90:5873-5877).

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA), MacVector and Assembler Version 12.7 (Macvector. 1939 High HouseRoad, Cary, N.C. USA 27519. Alignments using these programs can beperformed using the default parameters.

Greater than 90% sequence identity, or even 100% sequence identity,between the inhibitory RNA and the portion of the eukaryotic translationinitiation factor target gene is preferred. Alternatively, the duplexregion of the RNA may be defined functionally as a nucleotide sequencethat is capable of hybridizing with a portion of the target genetranscript under stringent conditions (e.g., 400 mM NaCl, 40 mM PIPES pH6.4, 1 mM EDTA, 60° C. hybridization for 12-16 hours; followed bywashing). The length of the substantially identical double-strandednucleotide sequences may be at least about 18, 19, 21, 25, 50, 100, 200,300, 400, 491, 500, or 510 bases. In a preferred embodiment, the lengthof the constructed double-stranded nucleotide sequence is approximatelyfrom about 18 to about 510 nucleotides in length.

The dsRNA construct disclosed herein may optionally comprise a singlestranded overhang at either or both ends. The double-stranded structuremay be formed by a single self-complementary RNA strand (i.e. forming ahairpin loop) or two complementary RNA strands. RNA duplex formation maybe initiated either inside or outside the cell. When the dsRNA of theinvention forms a hairpin loop, it may optionally comprise an intron, asset forth in U.S. 2003/0180945A1 or a nucleotide spacer, which is astretch of sequence between the complementary RNA strands to stabilizethe hairpin transgene in cells. Methods for making various dsRNAmolecules are set forth, for example, in WO 99/53050 and in U.S. Pat.No. 6,506,559. The RNA may be introduced in an amount that allowsdelivery of at least one copy per cell. Expression of higher doses ofdouble-stranded construct may yield more effective RNA viral plantresistance.

While the examples provided wherein describe dsRNA constructs targetingeukaryotic initiation factor genes eIF(iso)4E-2 and eIF4G, (GenBankAccession Nos. Q03389 and EF190330), it is contemplated that when readin conjunction with the teaching disclosed herein, the construction ofother dsRNA constructs targeting other eukaryotic initiation factorcomplex members which in turn reduce or eliminate viral reproduction andreplication are disclosed herein.

Additionally it is contemplated that the disclosure herein would teachthe transfer of a nucleic acid fragment into the genome of a hostorganism, resulting in genetically stable inheritance, particularly in aplant. Host organisms containing the transformed nucleic acid fragmentsare referred to as “transgenic” organisms. Examples of methods of planttransformation include Agrobacterium-mediated transformation (De Blaereet al. 1987. Meth. Enzymol. 143: 277) and biolistic particle delivery or“gene gun” transformation technology (Klein et al. 1987. Nature (London)327: 70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference).Thus, isolated polynucleotides of the present invention can beincorporated into recombinant constructs, typically DNA constructs,capable of introduction into and replication in a host cell. Such aconstruct can be a vector that includes a replication system andsequences that are capable of transcription and translation of apolypeptide-encoding sequence in a given host cell. A number of vectorssuitable for stable transformation of plant cells or for theestablishment of transgenic plants have been described in, e.g., Pouwelset al. 1985. Supp. 1987. Cloning Vectors: A Laboratory Manual; Weissbachand Weissbach. 1989. Methods for Plant Molecular Biology, AcademicPress, New York; and Flevin et al. 1990. Plant Molecular Biology Manual,Kluwer Academic Publishers, Boston. Typically, plant expression vectorsinclude, for example, one or more cloned plant genes under thetranscriptional control of 5′ and 3′ regulatory sequences and a dominantselectable marker. Such plant expression vectors also can contain apromoter regulatory region (e.g., a regulatory region controllinginducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and/or apolyadenylation signal.

It is to be understood that as used herein the term “transgenic”includes any cell, cell line, callus, tissue, plant part, or plant thegenotype of which has been altered by the presence of a heterologousnucleic acid including those transgenics initially so altered as well asthose created by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

The teachings disclosed herein also teach generating a non-transgenicwheat plant having the genes for eIF(iso)4E-2 or eIF4G silenced throughthe use of chemical mutagens such as ethyl methanesulfonate (EMS),radiation based mutagenesis such as fast neutron, or site directedmutagenesis, and targeted editing such as zinc finger nucleases, TALEN,or CRISPR technologies. An example would be to expose wheat seeds to0.6% EMS. M0 seeds would be grown to maturity and allowed to selfpollinate. Plants would be screened at the M2 generation for mutationsin the eIF(iso)4E-2. DNA from lines would be pooled and TILLING would beused to identify mutant lines. Lines would be screened for resistance toWSMV and TriMV. Resistant lines would be used as germplasm for cropimprovement. A second approach would be to use targeted mutations toalter the binding site of an initiation factor that binds to a bindingprotein to prevent interactions with viral proteins, thus preventingvirus replication.

In an embodiment of the invention, targeted cleavage events can be used,for example, to induce targeted mutagenesis, induce targeted deletionsof cellular DNA sequences, and facilitate targeted recombination andintegration at a predetermined chromosomal ppTAC or ppeTAC locus.Techniques of nucleotide editing can be found for example, Urnov et al.(2010) Nature 435(7042):646-51; United States Patent Publications20030232410; 20050208489; 20050026157; 20050064474; 20060188987;20090263900; 20090117617; 20100047805; 20110207221; 20110301073;2011089775; 20110239315; 20110145940; and International Publication WO2007/014275, the disclosures of which are incorporated by reference intheir entireties for all purposes. Cleavage can occur through the use ofspecific nucleases such as engineered zinc finger nucleases (ZFN),transcription-activator like effector nucleases (TALENs), or using theCRISPR/Cas system with an engineered crRNA/tracr RNA (‘single guideRNA’) to guide specific cleavage. U.S. Patent Publication No.20080182332 describes the use of non-canonical zinc finger nucleases(ZFNs) for targeted modification of plant genomes; U.S. PatentPublication No. 20090205083 describes ZFN-mediated targeted modificationof a plant EPSPS locus; U.S. Patent Publication No. 20100199389describes targeted modification of a plant Zp15 locus and U.S. PatentPublication No. 20110167521 describes targeted modification of plantgenes involved in fatty acid biosynthesis. In addition, Moehle et al.(2007) Proc. Natl. Acad, Sci. USA 104(9):3055-3060 describes usingdesigned ZFNs for targeted gene addition at a specified locus. U.S.Patent Publication 20110041195 describes methods of making homozygousdiploid organisms.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, andprogeny of same. Parts of transgenic plants are to be understood withinthe scope of the invention to comprise, for example, plant cells,protoplasts, tissues, callus, embryos as well as flowers, stems, fruits,leaves, roots originating in transgenic plants or their progenypreviously transformed with a DNA molecule of the invention andtherefore consisting at least in part of transgenic cells, are also anobject of the present invention.

EXAMPLE 1 Cloning of eIF(iso)4E-2 (SEQ. ID. NO: 2)

A search term of “eIF4F” was used to identify a partial cDNA sequencefrom the TIGR wheat EST database (http:\jcvi.org\wheat\ wheat gene indexterm TC383303). That sequence was used in a BLASTX search to find thefull length Triticum aestivum L. EIF(iso)4E-2 amino acid sequence inGenBank (Accession number Q03389). The nucleotide sequence of Q03389 wasused to design the primers used to clone a portion of eIF(iso)4E-2.Forward CACCCGCAAATGGAGGCAAATGGACTGT (SEQ. ID. NO: 5) and ReverseTCCACCTCTGCTTGGTTTCTGACT (SEQ. ID. NO: 6) primers were used to amplify a298 fragment of eIF(iso)4E-2 from cDNA from the cultivar Bobwhitecorresponding to nucleotides 318-615.

CACC was added to the 5′ end of the forward primer for directionalcloning of the PCR fragment into the entry vector pENTER-D/TOPO (LifeTechnologies) which carries two recombination sites, attL1 and attL2.The CP fragment was independently cloned into pANDA mini vector (Mikiand Shimamoto, 2003; FIG. 1A, FIG. 1B) by means of homologousrecombination via LR clonase (Invitrogen, Carlsbad, Calif.).

EXAMPLE 2 Generation of Silenced eIF(iso)4E-2 Transgenic WheatConferring Resistance to Viruses

The construct was bombarded in five independent biolistic experimentsusing approximately 900 independent wheat embryos. Embryogenic calli wastransferred into glufosinate selection and regeneration cycles, wheremorphological differentiation occurred normally. One hundred andnineteen plants were generated under glufosinate selection, andtransferred to soil. Plants were tested for glufosinate resistance and15 herbicide resistant plants were identified. PCR analyses of theseplants identified three with the hairpin construct. Lines were selffertilized and tested in the T3 generation. The majority of the linesderived from single seed descent that contained the gene of interest(GOI) and were expressing were resistant to Wheat streak mosaic virus(WSMV) and Triticum Mosaic Virus (TriMV) both individually and duringco-infection (Table 1). Seed from the T2 generation were bulked fortesting with Soilbourne mosaic virus (SbWMV). Soil infected with SbWMVwas collected and used to grow plants. Most plants that had the GOI andwere expressing the GOI were found to be resistant to the virus (Table2). Bulked T₂ seed was also used for testing resistance to Barley yellowdwarf virus (BYDV). A viruliferous aphid colony containing three strainsof BYDV, -SGV, -MAV and PAV were used. Ten viruliferous aphids wereplaced individually on each plant and allowed time to infect. Aphidswere then destroyed. Plants were allowed to recover. Most plants thatcontained the GOI and were expressing were found to be resistant (Table3).

TABLE 1 Transgenic wheat plant expressing silenced eIF(iso)4E-2co-infected with WSMV and TriMV. Transgenic plants were T3 generationderived by single seed selection. Transgenic Bobwhite controls werelines expressing only the bar gene for biolophos resistance. T3 GOI +and Resistant to Resistant to eIF4(iso)4E-2 Total Plants Expressing WSMVTriMV 1550A 50 47 45 45 1822A 53 45 40 40 1814A 54 52 43 43 Transgenic24 0 0 0 Bobwhite w/o GOI

TABLE 2 Transgenic wheat plant expressing silenced eIF(iso)4E-2 infectedwith SbWMV. Transgenic plants were T3 generation bulk derived T2progeny. Transgenic Bobwhite controls were lines expressing only the bargene for biolophos resistance. T3 GOI + and Resistant to eIF4(iso)4E-2Total Plants Expressing SbWMV 1550A 75 32 26 1822A 70 28 24 1814A 73 3024 Transgenic 17 0 0 Bobwhite w/o GOI

TABLE 3 Transgenic wheat plant expressing silenced eIF(iso)4E-2 infectedwith BYDV. Transgenic plants were T3 generation bulk derived T2 progeny.Transgenic Bobwhite controls were lines expressing only the bar gene forbiolophos resistance. T3 GOI + and Resistant to eIF4(iso)4E-2 TotalPlants Expressing BYDV 1550A 16 3 3 1822A 10 7 6 1814A 9 6 6 Transgenic2 0 0 Bobwhite w/o GOI

EXAMPLE 3 Cloning of eIF4G (SEQ: ID. NO. 4)

A search of “Triticum aestivum eIF4G” was used to identify the wheateIF4G cDNA (EF190330) sequence from GenBank and used to design primersused to clone a portion of wheat eIF4G. The primers used were ForwardCACCTCAGCAGCACCATTGGTATCTCCA (SEQ. ID. NO: 7) and ReverseGCTCGGAGCATTCAACTCCTCAA (SEQ. ID. NO: 8). The primers amplified afragment of a 517 bp from regions 3479-3993 of the eIF4G sequence(EF190330). The fragment was amplified from cDNA from the cultivarBobwhite.

CACC was added to the 5′ end of the forward primer for directionalcloning of the PCR fragment into the entry vector pENTER-D/TOPO (LifeTechnologies) which carries two recombination sites, attL1 and attL2.The CP fragment was independently cloned into pANDA mini vector (Mikiand Shimamoto, 2003; FIG. 1A, 1B) by means of homologous recombinationvia LR clonase (Invitrogen, Carlsbad, Calif.).

EXAMPLE 4 Generation of Silenced eIF4G Transgenic Wheat ConferringResistance to Viruses

A fragment of 517 bp from the host eukaryotic translation initiationfactor G (GenBank Accession #EF190330.1) was cloned in pANDA mini andco-bombarded with pAHC20 in five independent biolistic experiments usingapproximately 900 wheat embryos. Embryogenic calli was transferred ontoglufosinate selection. Seventy-two plants were generated underglufosinate selection and transferred to soil. PCR analyses confirmedthe presence of the eIF4G hairpin construct in three lines of thebar-positive plants. Lines were self fertilized through the T3generation. At the T3 generation, plants were inoculated with eitherWSMV or Triticum mosaic virus (TriMV) at the 2-3 leaf stage. Fourteendays later, plants were inoculated again to insure infection. Twenty-onedays after the second inoculation, plants were scored for viral symptomsand compared to the nontransformed Bobwhite susceptible control. Leafsamples were also taken and used for ELISA to determine the presence ofviral antigen. The majority of the lines that contained the gene ofinterest (GOI) were resistant to WSMV and TriMV both individually andduring co-infection (Table 4). Seed from the T2 generation were bulkedfor testing with Soilbourne mosaic virus (SbWMV). Soil infected withSbWMV was collected and used to grow plants. Most plants that had theGOI and were expressing the GOI were found to be resistant to the virus(Table 5). Bulked T2 seed was also used for testing resistance to Barleyyellow dwarf virus (BYDV). A viruliferous aphid colony containing threestrains of BYDV, -SGV, -MAV and PAV were used. Ten viruliferous aphidswere placed individually on each plant and allowed time to infect.Aphids were then destroyed. Plants were allowed to recover. Most plantsthat contained the GOI and were expressing were found to be resistant(Table 6). These results indicate the eIF4G hairpin construct providesresistance to the two potyviruses, the luteovirus and the furovirus.

TABLE 4 Transgenic wheat plant expressing silenced eIF4G co-infectedwith WSMV and TriMV. Transgenic plants were T3 generation derived bysingle seed selection. Transgenic Bobwhite controls were linesexpressing only the bar gene for biolophos resistance. T3 GOI + andResistant to Resistant to eIF4G Total Plants Expressing WSMV TriMV 1673A50 47 47 47 1742A 51 48 48 48 1755A 50 48 48 48 1830A 46 52 42 42Transgenic 24 0 0 0 Bobwhite w/o GOI

TABLE 5 Transgenic wheat plant expressing silenced eIF4G infected withSbWMV. Transgenic plants were T3 generation bulk derived T2 progeny.Transgenic Bobwhite controls were lines expressing only the bar gene forbiolophos resistance. T3 GOI + and Resistant to eIF4G Total PlantsExpressing SbWMV 1673A 10 6 6 1742A 29 19 15 1755A 20 12 9 1830A 12 7 7Transgenic 11 0 0 Bobwhite w/o GOI

TABLE 6 Transgenic wheat plant expressing silenced eIF4G infected withBYDV. Transgenic plants were T3 generation bulk derived T2 progeny.Transgenic Bobwhite controls were lines expressing only the bar gene forbiolophos resistance. T3 GOI + and Resistant to eIF4G Total PlantsExpressing BYDV 1673A 10 4 3 1742A 5 1 1 1755A 7 2 2 1830A 5 3 3Transgenic 7 0 0 Bobwhite w/o GOI

While the invention has been described with reference to details of theillustrated embodiment, these details are not intended to limit thescope of the invention as defined in the appended claims. All citedreferences and published patent applications cited in this applicationare incorporated herein by reference. The embodiment of the invention inwhich exclusive property or privilege is claimed is defined as follows:

1. A non-transgenic plant comprising an eIF(iso)4E-2 allele in itsgenome, whereby the eIF(iso)4E-2 allele is an allele which encodes aprotein comprising the amino acid sequence of SEQ. ID. NO: 2,characterized in that said eIF(iso)4E-2 allele comprises one or moremutations in its nucleotide sequence and whereby as a result of said oneor more mutations the plant comprising said mutant allele in its genomehas significant resistance to RNA viruses compared to a plant comprisinga wild type eIF(iso)4E-2 allele in its genome.
 2. The non-transgenicplant of claim 1 wherein the plant is resistant to RNA plant virusesselected from the group consisting of Potyviruses, Luteoviruses, andFuroviruses.
 3. The non-transgenic plant of claim 1 wherein the plant isresistant to Wheat streak mosaic virus.
 4. The non-transgenic plant ofclaim 1 wherein the plant is resistant to Triticum mosaic virus.
 5. Themethod of claim 1 wherein the plant is resistant to Soil bourne mosaicvirus.
 6. The method of claim 1 wherein the plant is resistant to Barleyyellow dwarf virus.
 7. The seed of a non-transgenic plant produced bythe method of claim
 1. 8. A non-transgenic plant comprising an eIF4Gallele in its genome, whereby the eIF4G allele is an allele whichencodes a protein comprising the amino acid sequence of SEQ. ID. NO: 4,characterized in that said eIF4G allele comprises one or more mutationsin its nucleotide sequence and whereby as a result of said one or moremutations the plant comprising said mutant allele in its genome hassignificant resistance to RNA viruses compared to a plant comprising awild type eIF4G allele in its genome.
 9. The non-transgenic plant ofclaim 8 wherein the plant is resistant to RNA plant viruses selectedfrom the group consisting of Potyviruses, Luteoviruses, and Furoviruses.10. The non-transgenic plant of claim 8 wherein the plant is resistantto Wheat streak mosaic virus.
 11. The non-transgenic plant of claim 8wherein the plant is resistant to Triticum mosaic virus.
 12. The methodof claim 8 wherein the plant is resistant to Soil bourne mosaic virus.13. The method of claim 8 wherein the plant is resistant to Barleyyellow dwarf virus.
 14. The seed of a non-transgenic wheat plantproduced by the method of claim 8.